Method for sequencing both strands of a double stranded DNA in a single sequencing reaction

ABSTRACT

A method is presented which uses a unique opposite strand joining strategy during PCR of an original DNA to generate a product which, when sequenced with a single sequencing primer yields the sequence of both strands of the original DNA. The PCR primers include 1) a modified oligomer corresponding to the 5′ end of a first strand of the DNA to be amplified wherein said modified oligomer includes the reverse complementary sequence to a sequence within said first strand of DNA and a specific PCR priming sequence which will specifically hybridize to a portion of the DNA to be amplified and 2) a second oligomer corresponding to the 5′ end of the second strand of the DNA to be amplified and which contains the priming sequence for the second strand of the DNA and will specifically hybridize to a portion of the DNA to be amplified. During PCR an intermediate product is formed where one end of one strand loops around to hybridize to its complement on the same strand. This results in a hairpin structure which elongates using its own strand as a template to form a double sized product that contains the sequence of both original strands. Upon denaturation this yields single strands with the single strands having the sequence of both of the original strands included in tandem. Sequencing these single strands using a single primer, e.g., a primer complementary to the second oligomer, yields the sequences of both strands of the DNA of interest.

This application is a divisional of Ser. No. 09/472,877 filed Dec. 28,1999 which in turn is a divisional application of Ser. No. 08/925,277filed Sep. 8, 1997 and which issued as U.S. Pat. No. 6,087,099 and whichare incorporated herein by reference.

BACKGROUND OF THE INVENTION

Sequencing of nucleic acids is an extremely important and widely usedtechnique. It is used for a variety of purposes. One such purpose is toidentify whether mutations within genes of known sequence are present ina sample of DNA taken from a person. This is especially important indiagnosing whether the person may have a disease which is known to beassociated with specific mutations in the gene being analyzed. When thistype of testing is performed, it is common to sequence both strands ofDNA to minimize any errors which may occur in the sequencing. To date,when sequencing both strands by the Sanger dideoxy method there has beena requirement to use one primer to sequence the sense strand and asecond primer to sequence the antisense strand of the double-strandedDNA. The two strands have been sequenced in separate sets of reactions.The present invention is a technique by which both strands of DNA aresequenced in a single set of reactions using only a single primer. Thismethod allows one to use fewer reactions for obtaining the data. This isespecially important for laboratories which will be processing manysamples. The use of fewer reactions will decrease the cost of analysis.

DNA sequencing methods were developed during the 1970s by Maxam andGilbert (1977) and by Sanger (1977). The Sanger method which usesdideoxy nucleotides to terminate newly synthesized DNA strands is mostcommonly used and has been adapted such that it can be used withfluorescent markers rather than radioactivity. One variation is atechnique called cycle sequencing in which DNA sequencing is combinedwith polymerase chain reaction (PCR). Chadwick et al. (1996) teach avariation of cycle sequencing in which mutant Taq DNA polymerase isutilized.

The polymerase chain reaction itself is only one of a number ofdifferent methods now available for amplifying nucleic acids. Some ofthe other methods include ligase chain reaction (Wu and Wallace, 1989),Strand Displacement Amplification (SDA) (Walker, U.S. Pat. No. 5,455,166(1995); Walker et al., 1992), thermophilic SDA (Spargo et al., 1996),and 3SR or NASBA (Compton, 1991; Fahy et al., 1991).

The instant invention is a method of using a specially designed oligomerwhich contains a reverse complement sequence along with a standardprimer during PCR. This generates a double stranded DNA product suchthat when it is denatured one end of the resulting single stranded DNAloops around to form an intrastrand stem-loop structure. This structureis then elongated thereby producing a double-stranded DNA but whereinthe two strands are joined by a loop. This method is referred to asopposite strand joining PCR. When denatured this product forms asingle-stranded DNA which contains both strands of the original DNA.When this resulting single-stranded DNA is sequenced it yields thesequence of both strands of the original double-stranded DNA.

A similar stem-loop DNA structure was used as a template for PCRamplification by Jones et al. (1992). The Jones et al. referencedescribes a “panhandle PCR” method. This technique introduced aself-complementary portion into the target DNA strand by ligation. Thegoal of panhandle PCR is to amplify unknown sequence by generating astem loop template structure for PCR whereas one of the goals ofopposite strand joining PCR is to amplify known sequence by generating astem-loop structure during PCR and then sequencing both strands of thelonger product in one sequencing reaction. Another use for oppositestrand joining PCR is in denaturing gradient gel electrophoresistechniques wherein the use of this technique can form a covalentlybonded hairpin loop which can replace the use of a GC clamp. Yet anotheruse for opposite strand joining PCR is simply the use of the methodeffectively to join together the two strands of any double stranded DNAinto a single strand of DNA for any desired purpose.

SUMMARY OF THE INVENTION

A method is presented which uses a unique opposite strand joiningstrategy during PCR of an original DNA to generate a product which, whensequenced with a single sequencing primer yields the sequence of bothstrands of the original DNA. The PCR primers include 1) a modifiedoligomer corresponding to the 5′ end of a first strand of the DNA to beamplified wherein said modified oligomer includes the reversecomplementary sequence to a sequence within said first strand of DNA anda specific PCR priming sequence which will specifically hybridize to aportion of the DNA to be amplified and 2) a second oligomercorresponding to the 5′ end of the second strand of the DNA to beamplified and which contains the priming sequence for the second strandof the DNA and will specifically hybridize to a portion of the DNA to beamplified. During PCR an intermediate product is formed where one end ofone strand loops around to hybridize to its complement on the samestrand. This results in a hairpin structure which elongates using itsown strand as a template to form a double sized product that containsthe sequence of both original strands. Upon denaturation this yields asingle strand having the sequence of both of the original strandsincluded in tandem. Sequencing these single strands using a singleprimer, e.g., a primer complementary to the second oligomer, yields thesequences of both strands of the DNA of interest.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D show the primer design used in the Example. FIG. 1A showsthe sequence of primer 19R which consists of the −28M13 reverse DETprimer sequence (shown in bold) which is 5′ to the gene specificsequence G2′. FIG. 1B shows the sequence of primer 19F which consists ofthe −40M13 forward DET sequence (shown in bold) which is 5′ to the genespecific sequence G1. FIG. 1C shows the sequence of the opposite strandjoining primer 19XF which consists of a short reverse complementedgenomic sequence C′ (shown in bold) which is 5′ to the gene specificsequence G1 used in primer 19F. FIG. 1D shows the genomic sequence inthe region of the opposite strand joining primer. The gene specificsequence G1 (shown in nonbolded upper case letters) used in both the 19Fand 19XF primers is 5′ of sequence C (shown in bold upper case letters).It is this genomic region C which is reverse complemented (and thereforecalled C′) and placed 5′ to the gene specific sequence G1 in theopposite strand joining primer 19XF.

FIGS. 2A-2F illustrate the opposite strand joining strategy. Throughoutthese figures, all the strands labeled  SEQ are substrates for dyeprimer sequencing.

FIG. 2A shows genomic DNA in the region of exon 19. This is shown asfour sections on each strand with one strand having G1, C, the exon 19containing region, and G2 and the opposite strand being designated withprimes, e.g., G1′, C′, etc. Region C is the portion which iscomplementary to a portion of primer 19XF and which will hybridize withthe appended primer so that there is intrastrand hybridization forming aloop and a double-stranded region of DNA. The exon 19 region is theregion of interest to be sequenced.

FIG. 2B shows the product obtained when the DNA shown in FIG. 2A issubjected to PCR with primers 19XF and 19R. The major amplified productis similar to the DNA of FIG. 2A but has had a C′/C tail added at oneend and an M13R/M13R′ tail added at the other end.

FIG. 2C shows the exon 19′ strand of FIG. 2B following denaturation.This illustrates that intrastrand binding occurs forming a loop with theC tail hybridizing to the C′ portion of the strand. This product is asubstrate for dye terminator sequencing in the absence of primer.

FIG. 2D illustrates the product formed after elongating the DNA shown inFIG. 2C. The product, which upon denaturation is a single-strandedmolecule, contains both strands of exon 19, i.e., it contains both exon19 and exon 19′ in a tandem arrangement (separated by C, G1′ and C′).

FIG. 2E illustrates the product formed when the DNA of FIG. 2D undergoesanother cycle of PCR using primer 19R. Note that the DNA shown in FIG.2E is palindromic except for the very central G1/G1′ region andsequencing both strands yields the identical sequence except for theG1/G1′ region (which is not of interest). G1 and G1′ are complementaryand are of equal length and therefore the sequence obtained from bothstrands using a single primer is identical throughout except for thecentral G1/G1′ section which will not interfere with the reading of therest of the sequence. The full length products formed and shown in FIG.2E can then be used in further rounds of PCR using primer 19R.

FIG. 2F illustrates the products formed when the DNA of FIG. 2Dundergoes another cycle of PCR using primers 19XF and 19R. The productshown is a result of primer 19XF priming this cycle on the FIG. 2D DNA.Each strand of this product can reenter the cycle of steps at step A(short strand) or step D (long strand).

FIGS. 3A-3D show the sequence comparison of both strands of the BRCAlexon 19 obtained from a single sequencing lane.

FIG. 3A shows the sequence of exon 19′ of the products B, D, E and F(shown in FIG. 2) amplified by the primers 19XF and 19R for whichsequencing the −28M13 reverse primer was used.

FIG. 3B shows the sequence of exon 19 of the products B, D, E and F(shown in FIG. 2) amplified by the primers 19XF and 19R for whichsequencing the −28M13 reverse primer was used.

FIG. 3C shows the sequence of exon 19′ of the product amplified by thestandard primers 19F and 19R gene for which sequencing the −28M13reverse primer was used.

FIG. 3D shows the sequence of exon 19 of the product amplified by thestandard primers 19F and 19R for which sequencing the −40M13 forwardprimer was used.

DESCRIPTION OF THE INVENTION

The present invention is directed to sequencing both strands of adouble-stranded DNA molecule by using only a single set of labeledprimers rather than using two sets of labeled primers as is doneconventionally. The ability to sequence both strands using a single setof reactions is more efficient and less expensive. The method isespecially appropriate for sequencing both strands of shorter pieces ofDNA such that one strand of a DNA of double length could be sequenced ina single sequencing run by conventional methods.

The present invention is also especially suitable for use in clinicallaboratories which will be sequencing large numbers of samples of genesof known sequence to determine whether the samples contain mutations. Asan example, a diagnostic test for breast cancer (BRACAnalysis™) involvescomplete PCR and sequencing of the coding sequences and proximal intronsof both alleles of a patient's BRCA1 and BRCA2 genes in order to findany deleterious mutations. To ensure high quality and consistency, thediagnostic test is completely automated. A total of 35 amplicons forBRCA1 and 47 amplicons for BRCA2 are sequenced. The PCR productssequenced during the standard BRACAnalysis™ are amplified with 5′ M13tailed gene specific primers. Following amplification, the productscontain the M13 tail sequences at their ends. During sequencing bothstrands of the amplified products are sequenced in two separatereactions. The sequencing reactions are ethanol precipitated andresolved in two separate lanes on an Applied Biosystems 377 sequencinggel. The sequences obtained are analyzed for the presence of mutationsand polymorphisms.

The instant invention involves a novel concept to sequence both strandsof the amplified products in one sequencing reaction. The sequenceobtained for both the DNA strands is present in a single lane of asequencing gel. The Example below, which is not intended to limit theinvention in any manner, describes use of this method for exon 19 in theBRCA1 gene. The tailed genomic primer 19R (see FIG. 1), developed forthe standard BRACAnalysis™ test, in combination with the unique oppositestrand joining primer 19XF is used for PCR. During PCR an intermediateturnaround strand is formed where the 3′ end loops around and hybridizesto a complementary region on the same strand thus generating a stem loopstructure. Elongation of this stem loop structure at the 3′ end resultsin the formation of a longer product which contains the sequence of bothstrands. In a complex multiamplicon test such as BRACAnalysis™,application of this technique to each suitable amplicon willsubstantially reduce the number of sequencing reactions and the numberof sequencing gel lanes used, making the test more cost efficient.

The method disclosed here is designated “opposite strand joining PCR”.It uses an opposite strand joining primer during PCR to generate aturnaround structure resulting in the formation of a double size DNAstrand (FIGS. 2C and 2D). Combination of the opposite strand joiningprimer (19XF in FIG. 1C) and the 19R primer (FIG. 1A) was used duringPCR. PCR with these two primers results in the formation of adouble-stranded intermediate product of which one of the strandscontaining the M13 tail at the 3′ end can be sequenced using the −28M13reverse sequencing primer (FIG. 2B). The 3′ end of the other strand canturn around to form a stem loop structure by intra-strand annealingwhere the 3′ end hybridizes to the complementary sequence on the samestrand (FIG. 2C). This 3′ end can then be used as a primer for the samestrand and elongate to form the double size product (FIG. 2D). Duringthe next PCR cycle, this longer product denatures and anneals with the19XF and 19R primers which elongate. This results in the formation ofproducts as shown in FIGS. 2E and 2F. These products are templates forprimer annealing and elongation during the next PCR cycles.

To verify the formation of the turnaround intermediate structure in FIG.2C, dye terminator sequencing was performed for the products amplifiedby the primers 19XF and 19R. The turned around 3′ end (FIG. 2C) acts asa primer and elongates using the same strand as a template during dyeterminator sequencing. Sequence of only one strand was observed by dyeterminator sequencing (data not shown). Thus, PCR amplification with anopposite strand joining primer enables dye terminator sequencing to becarried out without a primer.

The technique of opposite strand joining PCR is useful for modifyingdenaturing gradient gel electrophoresis (DGGE), a technique used formutation screening. Single base changes in the DNA have been detected byDGGE using a GC clamp attached to one end of the amplified product(Fischer et al., 1983; Myers et al., 1985a; Myers et al., 1985b).Addition of a GC clamp at the end of the PCR product using a modifiedprimer creates a high melting temperature region making it possible todetect base changes in the rest of the strand. The GC clamp can bereplaced by a covalently bonded hairpin loop by designing an oppositestrand joining primer. The region of interest is amplified using thecombination of the opposite strand joining primer designed at one endand a conventional primer at the other end of the region.

DEFINITIONS

A “primer” is an oligomer which will hybridize to a strand of nucleicacid and which can be extended or elongated by the addition ofnucleotides to form a nucleic acid strand of complementary sequence tothe strand of nucleic acid to which the primer is hybridized. Oneexample of such a reaction is the polymerase chain reaction in which twoprimers are used wherein one primer is complementary to the 5′ end ofnucleic acid to be amplified and a second primer is complementary to the3′ end of nucleic acid to be amplified and further wherein one primer iscomplementary to the sense strand and the other primer is complementaryto the antisense strand. Another example is a sequencing reactionwherein the DNA to be sequenced is made single-stranded and a primer isadded which primer is complementary to a portion of one of the singlestrands of DNA and is elongated.

A “single primer” means a primer comprising a single nucleotidesequence. The phrase “single primer” may encompass more than only oneprimer. It encompasses, e.g., four distinct primers which all haveidentical nucleotide sequences but which are labeled with four distinctmarkers such as four different fluors wherein each primer moleculecomprises one of the four fluors. Each of these four primers may be usedseparately in sequencing reactions, yet they are together considered tobe a single primer. Alternatively, a single primer may in fact representonly one primer which is identical in all cases, such as will occur whensequencing using a radioactively labeled primer or radioactively labeleddNTPs or when performing dye terminator sequencing, but the definitionis not so limited for purposes of the present disclosure.

A “single set of sequencing reactions” refers to the reactions necessaryto sequence a single strand of DNA. Commonly a single set of sequencingreactions will consist of four separate reactions which later are eitherrun on four lanes of a gel if a radioactive label is used or are mixedtogether and run on a single lane of a gel if fluorescent labels areused.

A “reverse complementary sequence of nucleotides” refers to a sequenceof nucleotides within a strand of DNA which is complementary to anothersequence of nucleotides within the same strand of DNA but in the reverseorder such that when the single strand folds back upon itself thereverse complementary sequence of nucleotides can hybridize to itscomplementary sequence within the same strand thereby yielding a hairpinstructure.

“Effectively to join” together two strands of a double-stranded DNA intoa single-stranded DNA means to use a method which does not actually jointhe two existing strands of double-stranded DNA together but which hasthe same effect as so doing for a portion of the double-stranded DNA.The original double-stranded DNA is amplified and the newly formed DNAundergoes steps to yield a single-stranded DNA which includes the samesequences as found in the two strands of a portion of the amplifiedregion of the double-stranded DNA. The result is that, although the twostrands of the original double-stranded DNA are not themselves joinedtogether, the effect is the same as having done so for a portion of theoriginal double-stranded DNA.

EXAMPLE A. Primer Design

The primers 19F and 19R (see FIGS. 1A and 1B) are the primers for exon19 of the BRCA1 gene used in the standard BRACAnalysiS™ diagnosticassay. These primers contain the gene specific region and the −40M13forward or −28M13 reverse DYEnamic energy transfer (DET) primer sequencefrom Amersham Life Science at its 5′ end.

The opposite strand joining primer, 19XF (FIG. 1C), for exon 19 wasdesigned as follows. This primer contains the same gene specificsequence G1 as the 19F primer but the sequence at the 5′ end contains a9 basepair reverse complemented genomic sequence (C′). The genomicsequence C which corresponds to C′ is present 3′ to the 19F genespecific sequence G1 in the genomic DNA (FIG. 1D and FIG. 2A).

B. Polymerase Chain Reaction

Human genomic DNA was amplified by PCR using the primer 19R incombination with either primer 19F (for the linear product) or 19XF (forthe turnaround product). The reactions were carried out in a totalvolume of 9 μL and contained 20 ng DNA, 0.2 mM each dNTP, 0.5 unitsAmplitaq Gold DNA polymerase (from Perkin-Elmer), 10 mM Tris pH8.3, 50mM KCl, 1 mM EDTA, 6.5 mM MgCl₂, 10% sucrose and 0.01% Tween 20, 0.1 μMof primer 19R and either 0.1 μM of primer 19F or 0.4 μM primer 19XF,respectively. The reactions were layered with oil and then cycled in theDNA Engine Thermal cycler at 94° C. for 10 minutes followed by 36 cyclesof 96° C. for 20 seconds, 62° C. for 30 seconds and 72° C. for 60seconds. This was followed by 1 cycle at 72° C. for 60 seconds.

C. Sequencing

Dye primer sequencing reactions were carried out with half volume of1:10 diluted PCR products, 0.2 μM dideoxynucleotide/45 μMdeoxynucleotide mix, 80 nM Tris pH 9.5, 2% sucrose, 0.05% Triton X, 1 mMEDTA, 5 mM MgSO₄, 0.075 units Taq FS polymerase (Kalman et al., 1995;Tabor et al., 1995) and 0.04 μM −40M13 forward or −28M13 reverse DETprimers (Ju et al., 1995a; Ju et al., 1995b). The reactions were layeredwith oil and then cycled in the DNA Engine Thermal cycler for 32 cyclesat 96° C. for 20 seconds, 56° C. for 30 seconds and 72° C. for 60seconds, followed by one cycle at 72° C. for 60 seconds.

The product amplified by the primers 19F and 19R was sequenced in bothdirections using the −40M13 forward and the −28M13 reverse sequencingprimers. The products created by the primers 19XF and 19R were sequencedusing the −28M13 reverse sequencing primer. The four forward (orreverse) reactions were pooled and ethanol precipitated. The precipitatewas resuspended in 50% formarnide, 50 mM EDTA, denatured and loaded onan Applied Biosystems 377 sequencing gel.

D. Results

FIGS. 2A-2F illustrate opposite strand joining PCR for exon 19 of BRCA1where one strand of the double stranded product shown in FIG. 2B, theturnaround product shown in FIG. 2D and both strands of the productsshown in FIGS. 2E and 2F are substrates for dye primer sequencing usingthe −28M13 reverse sequencing primer. The two strands of exon 19(strands 19 and 19′) are present on two different strands in the genomicDNA (FIG. 2A). When genomic DNA is amplified in the conventional mannerusing primers 19F and 19R, a double-stranded product is generated inwhich one strand contains the exon 19 strand and the opposite strandcontains the exon 19′ strand. In contrast to this conventional result,opposite strand joining PCR generates products shown in FIGS. 2D and 2Ein which the original exon 19 and exon 19′ strands are both containedwithin a single strand of DNA. The longer strand of the product shown inFIG. 2F also contains both the exon 19 and exon 19′ strands. Thesequence of exon 19 and exon 19′ in these products can be obtained byusing only one sequencing primer since the 19R primer has the −28M13sequence at its 5′ end whereas the 19XF primer has no M13 tail.

FIG. 3 illustrates the sequences obtained from the products amplified bythe primer combinations 19F with 19R (standard PCR) and 19XF with 19R(opposite strand joining PCR). Electropherograms A and B (FIGS. 3A and3B) represent the sequence in both directions for the products (FIGS.2B, 2D, 2E and 2F) amplified by the primers 19XF and 19R and sequencedwith the −28M13 reverse primer in a single reaction and in a single laneon a sequencing gel. Electropherograms C and D (FIGS. 3C and 3D)represent the sequence of the two strands for the product amplified bythe primers 19F and 19R and sequenced with the −40M13 forward or the−28M13 reverse primer in two separate reactions and in two lanes on asequencing gel. Comparison of the electropherograms A and C shows thesame sequence for the products generated by primers 19XF with 19R andfor the product generated by the primers 19F with 19R sequenced by thesame sequencing primer. Comparison of the electropherograms B and Dshows the same sequence for the products generated by primers 19XF with19R and for the product generated by primers 19F with 19R but sequencedby two different sequencing primers. Thus, from electropherograms A andB, the sequence of exon 19 of the BRCA1 gene can be read in bothdirections from a single set of sequencing reactions using only onesequencing primer.

In tests to optimize the above method of opposite strand joining PCR,various concentrations (0.0125 μM, 0.025 μM, 0.05 μM, 0.1 μM, 0.2 μM and0.4 μM) of the opposite strand joining primer, 19XF, in combination withvarious lengths (20 bases, 14 bases, 10 bases, 9 bases, 8 bases and 6bases) of the 5′ reverse complemented sequence while keeping theconcentration of the primer 19R at 0.1 μM. Equal sequence signalintensity values for both directions in the turnaround product were seenwhen the length of the 5′ reverse complemented sequence in the 19XFprimer was 9 bases and the concentration was 0.4 μM.

Those of skill in the art will realize that the Example is onlyillustrative and that many variations of the specific methods of theExample are possible. For example, one could perform a PCR reactionwhich adds the oligomers at the ends of the genomic DNA to produce thestructures shown in FIGS. 2E and 2F. These products can then besequenced with a single primer by means other than discussed in theExample. It is not necessary to use fluorescently labeled primers,radioactively labeled primers can be used instead, and it is unnecessaryto perform cycle sequencing, rather ordinary sequencing methods withoutcycling may be utilized. Similarly, there is no need to use the M13sequences as part of the primers as used in the Example. This could bereplaced by any other known sequence of DNA. Any gene sequence can beanalyzed in this manner and the use of BRCA 1 or BRCA2 was merelyintended to be illustrative. These variations and other variations willbe obvious to one of skill in the art and the disclosure is meant to beexemplary only and not inclusive of the means of performing theinvention.

LIST OF REFERENCES

Chadwick, R. B., M. P. Conrad, M. D. McGinnis, L. Johnston-Dow, S. L.Spurgeon and M. N. Kronick (1996). “Heterozygote and Mutation Detectionby Direct Automated Fluorescent DNA Sequencing Using a Mutant Taq DNAPolymerase.” BioTechniques 20:676-683.

Compton, J. (1991). “Nucleic acid sequence-based amplification.” Nature3:91-92.

Fahy, E., D. Y. Kwoh and T. R. Gingeras (1991). “Self-sustained sequencereplication (3SR): an isothermal transcription-based amplificationsystem alternative to PCR.” PCR Methods Appl. 1:25-33.

Fischer, S. G. and L. S. Lerman (1983). “DNA fragments differing bysingle base pair substitutions separated in denaturing gradient gels:Correspondence with melting theory.” Proc. Natl. Acad. Sci. USA80:1579-1583.

Jones, D. H. and S. C. Winistorfer (1992). “Sequence specific generationof a DNA panhandle permits PCR amplification of unknown flanking DNA.”Nucl. Acids. Res. 30:595-600.

Ju, J., 1. Kheterpal, J. R. Scherer, C. W. Fuller, A. N. Glazer and R.A. Mathies (1995a). “Design and synthesis of fluorescence energytransfer dye labeled primers and their application for DNA sequencingand analysis.” Annals of Biochemistry 231:131-140.

Ju, J., C. Ruan, C. W. Fuller, A. N. Glazer and R. A. Mathies (1995b).“Fluorescence energy transfer dye labeled primers for DNA sequencing andanalysis.” Proc. Natl. Acad Sci. USA 92:4347-4351.

Kalman, L. V., R. D. Abranson and D. H. Gelfand (1995). “ThermostableDNA polymerases with altered discrimination properties.” Genome ScienceTechnology 1:42.

Maxam, A. M. and W. Gilbert (1977). “A new method for sequencing DNA.”Proc. Natl. Acad. Sci. USA 74:560-564.

Myers, R. M., S. G. Fisher, T. Maniatis and L. S. Lerman (1985a).“Modification of the melting properties of duplex DNA by attachment of aGC-rich sequence as determined by denaturing gradient gelelectrophoresis.” Nucl. Acids Res. 13:3111-3129.

Myers, R. M., S. G. Fisher, L. S. Lerman and T. Maniatis (1985b).“Nearly all single base substitutions in DNA fragments joined toGC-clamp can be detected by denaturing gradient gel electrophoresis.Nucl. Acids Res. 13:3131-3145.

Sanger, F., S. Nicklen and A. R. Coulson (1977). “DNA sequencing withchain-terminating inhibitors.” Proc. Natl. Acad Sci. 2:5463-5467.

Spargo, C. A., M. S. Fraiser, M. van Cleve, D. J. Wright, C. M. Nycz, P.A. Spears and G. T. Walker (1996). “Detection of M. tuberculosis DNAusing thermophilic strand displacement amplification.” Mol. Cell. Probes10:247-256.

Tabor, S. and C. C. Richardson (1995). “A single residue in DNApolymerases of the Escherichia coli DNA polymerase I family is criticalfor distinguishing between deoxy- and dideoxyribonucleotides.” Proc.Natl. Acad. Sci. USA 9:6339-6343.

Walker, G. T. (1995). “Strand Displacement Amplification.” U.S. Pat. No.5,455,166.

Walker, G. T., M. S. Fraiser, J. L. Schram, M. C. Little, J. G. Nadeauand D. P. Malinowski (1992). “Strand displacement amplification—anisothermal, in vitro DNA amplification technique.” Nucl. Acids Res.20:1691-1696.

Wu, D. Y. and R. B. Wallace (1989). “The ligation amplification reaction(LAR)—amplification of specific DNA sequences using sequential rounds oftemplate-dependent ligation.” Genomics 4:560-569.

4 46 base pairs nucleic acid single linear other nucleic acid /desc =“Primer” not provided 1 AGGAAACAGC TATGACCATT GATCCTCATT ATCATGGAAAATTTGT 46 42 base pairs nucleic acid single linear other nucleic acid/desc = “Primer” not provided 2 GTTTTCCCAG TCACGACGGT CATTCTTCCTGTGCTCTTTT GT 42 33 base pairs nucleic acid single linear other nucleicacid /desc = “Primer” not provided 3 CAGCGATTCG TCATTCTTCC TGTGCTCTTTTGT 33 45 base pairs nucleic acid double linear DNA (genomic) NO notprovided 4 TCCTCTGTCA TTCTTCCTGT GCTCTTTTGT GAATCGCTGA CCTCT 45

What is claimed is:
 1. A method effectively to join both strands of adouble stranded DNA into a single strand of DNA, wherein saiddouble-stranded DNA consists of a first strand and a second strandwherein said single strand of DNA comprises at least a portion of saidfirst strand and said second strand, wherein said method comprises: a)amplifying said double-stranded DNA to form amplified DNA using a pairof primers wherein one of said primers comprises a reverse complementarysequence of nucleotides; b) denaturing said amplified DNA to formsingle-stranded DNA; c) allowing intrastrand hybridization of saidsingle-stranded DNA to occur; and d) elongating said intrastrandhybridized single-stranded DNA to yield panhandle DNA which upondenaturation yields a single-stranded DNA comprising sequence of saidboth strands of said double stranded DNA.